1.Add 750μl Reagent B (50mM Tris [pH8],10mM EDTA,100mM NaCl,1% [w/v] SDS)and 50μl 100mg/ml proteinase K to each spleen. 2.Incubate overnight at 56ºC. 3.Gently shake to dissociate tissue. 4.transfer 200μl to eppendorf tube. 5.Add 50μl 5M sodium perchlorate. 6.Incubate 25 mins 65ºC. 7.Add 200μl phenol/chloroform. 8.Mix by inversion for about 2 mins. 9.Spin 13000rpm for 2 mins. 10.Transfer aqueous phase (top layer)to new eppendorf. 14 Add 500μl ice cold 100% ethanol. 15.Spool DNA into 500μl TE 10:0.l (if no visible DNA see below). 16 Allow to resuspend at 4ºC overnight. If no visible DNA: 1.Spin 15 mins 13000rpm at ºC. 2.Remove liquid. 3.Add 500μl 70% ethanol. 4.Spin 5 mins 13000rpm at 4ºC. 5.Remove liquid and vac dry. 6.Add 500μl TE 10:0.1. |
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